This is a revised application submitted in response to RFA-HL-08-005 (Priority score = 191). Recent evidence suggests that red blood cells (RBCs) work in concert with endothelial cells to regulate tissue blood flow. Both RBCs (which are finely-tuned sensors of local O2 demand) and endothelial cells release mediators including nitric oxide (NO) that control vascular tone and thus blood flow and O2 delivery. Clinical studies suggest that RBC transfusions are associated with morbidity and mortality in recipients, and that these events are statistically associated with the use of aged RBC units and increased transfusion volumes. However, the occurrence of these events and their manifestations are variable, which has confounded efforts to study them. We hypothesize that donated RBCs stored under FDA-approved blood bank conditions have reduced capacity for NO synthesis and/or increased NO scavenging activity, both leading to reduced NO levels in vascular beds. Furthermore, a collection of other factors (reduced 2,3 DPG; endothelial dysfunction; elevated post-transfusion hematocrit levels) may further reduce NO bioavailability, leading to vasoconstriction, decreased blood flow, and reduced tissue O2 delivery. This hypothesis of insufficient NO bioavailability (INOBA) brings together previously unconnected data to suggest a common pathophysiologic mechanism linking RBC unit- and recipient-specific factors in the occurrence of morbidity/mortality in RBC transfusion recipients. This hypothesis not only accounts for the variability of adverse events following RBC transfusion, but also leads to a number of readily testable predictions that will be investigated in Aims 1-3 using detailed analyses of human transfusion recipients. PUBLIC HEALTH RELEVANCE: Red blood cell transfusions represent lifesaving therapies for anemic patients. However, transfusions are associated with adverse effects. These studies will seek to determine why adverse outcomes happen after transfusion, and develop ways to improve blood collection and storage to minimize these outcomes.